The invention discloses an establishment method for a brown mushroom three-level strain propagation system. The establishment method for the brown mushroom three-level strain propagation system comprises three-level strain propagation of stock cultures, liquid strains and cultispecies, wherein the formula of culture mediums of the stock cultures comprises, by weight (g/L), 600g of turfy soil, 20g of glucose, 100g of wheat grains, 1.5g of magnesium sulfate, 1g of monopotassium phosphate, 2g of peptone and 1000mL of water, the formula of the liquid strains comprises, by weight (g/L), 200g of potatoes, 20g of glucose, 2g of peptone, 2g of monopotassium phosphate, 1g of magnesium sulfate and 10.01g of VB, the pH (potential of hydrogen) is natural, and the cultispecies comprise, by weight (%), 92% of wheat grains, 5% of gypsum and 3% of turfy soil. Compared with a traditional solid state three-level strain, through using the propagation system, the propagation cycle is shortened by 38 days. The establishment method for the brown mushroom three-level strain propagation system is an optimum scheme, and has a certain guiding significance to the mass industrialized production of brown mushrooms.